The present invention relates to systems and methods for identifying, separating and isolating target analytes or impurities suspected of being present in a liquid sample but in quantities typically too low to detect using prior art mechanisms. The invention further relates to systems and methods for the detection and sequestration of target analytes based in part on volume reduction of the sample where such particles are believed to be present, coupled with the magnetic separation of such targeted analytes.
Separation techniques that are capable of identifying specific biological macromolecules, cells, and the like (collectively referred to as “biological particles”) are well-known in the art and used extensively for analytical and purification purposes in biological research, biomedical technology and diagnostic applications. In general, such separation techniques rely upon one or more physical and/or chemical properties of the target biological particle sought to be identified so as to capture or isolate the target particle at a fixed position or area. Among the properties that have been utilized to facilitate the identification and isolation of biological particles include density, size, hydrophobicity, electrical charge and surface chemical groups operative to react and bind with other materials and/or immunological agents. Exemplary of such techniques include: centrifugation, which can be used to separate cellular components based upon their relative density; liquid chromatography, which involves passing a sample over a packed column of particles that have a defined surface chemistry and/or porosity that are operative to interact and retain the target biological particles; and gel electrophoresis, which is operative to separate biological macromolecules via the application of an electric field, that in turn affects the mobility of such molecules to move through the gel in one or two dimensions based upon the charge-to-mass ratio of the macromolecule of interest.
Also frequently applied in separation techniques is microfluidics, which operate on the principle of manipulating and controlling fluids, usually in the range of microliters to picoliters, in networks of channels of different diameter, usually ranging from 5 to 500 μm. Such reduced dimensions are selectively chosen so that particles of a fluid sample, or particles suspended in the fluid sample, become comparable in size with the microfluidic apparatus itself. On such reduced scale, fluids are directed, mixed, separated or otherwise manipulated to attain multiplexing, automation, and high-throughput systems. Microfluidics can allow for the analysis and use of samples of much lesser volumes, as well as correspondingly lesser amounts of any chemicals and reagents utilized therewith, and have the capacity to both process and analyze samples with minor sample handling.
In addition to such techniques, there have further been utilized systems and methods for detecting biological macromolecules and cells of interest using magnetic particles that are operative to interact with an applied magnetic field. In a typical application, magnetic particles will carry a ligand on the surfaces thereof that enables the particle to bind specifically to a target biological macromolecule. In application, such magnetic particles are added to a sample, and allowed to bind with the macromolecule of interest, thereafter which a magnetic field is applied that enables the magnetic particles and the bound macromolecules of interest to be separated from the rest of the sample. The captured macromolecule of interest is then measured by detection, such as fluorescence-based emission, and can be used in conjunction with flow cytometric analysis.
References that are exemplary of the state of the art with respect to the separation of biological macromolecules, cells and the like are set forth in the following issued patent and published patent applications:                U.S. Pat. No. 6,479,302 B1, entitled METHOD FOR THE IMMUNOLOGICAL DETERMINATION OF AN ANALYTE, issued Nov. 12, 2002 to Bernd Dremel;        United States Published Patent Application No. 2006/0223178 A1, entitled DEVICES AND METHODS FOR MAGNETIC ENRICHMENT OF CELLS AND OTHER PARTICLES, published Oct. 5, 2006 to Barber et al.;        United States Published Patent Application No. 2007/0166835 A1, entitled MULTIPLEX ASSAYS USING MAGNETIC AND NON-MAGNETIC PARTICLES, published Jul. 19, 2007 to Mark N. Bobrow;        United States Published Patent Application No. 2010/0047766 A1, entitled ANALYTE MANIPULATION AND DETECTION, published Feb. 25, 2010 to Barrault et al.;        United States Published Patent Application No. 2010/0233675 A1, entitled ANALYTE MANIPULATION AND DETECTION, published Sep. 16, 2010 to Barrault et al.;        United States Published Patent Application No. 2012/0132593 A1, entitled SYSTEMS AND METHODS FOR MAGNETIC SEPARATION OF BIOLOGICAL MATERIALS, published May 31, 2012 to Murthy et al.;        United States Published Patent Application No. 2012/0270331 A1, entitled MICROFLUIDIC SYSTEM AND METHOD FOR AUTOMATED PROCESSING OF PARTICLES FROM BIOLOGICAL FLUID, published Oct. 25, 2012 to Achrol et al.; and        United States Published Patent Application No. 2016/0184737 A1, entitled NEW PROCESS AND SYSTEM FOR MAGNETIC SEPARATION, published Jun. 30, 2016 to Oscarsson et al.        
The teachings of all of the foregoing are expressly incorporated herein by reference.
Notwithstanding the general effectiveness of the aforementioned methodologies often times the target analyte of interest, despite being present in a sample, is in quantities too low to detect using such prior art techniques. In this regard, such methods are often unable to concentrate or enrich a sample sufficiently to allow analysis of rare components that may be present in the sample. In addition, such methodologies can result in unacceptable losses of rare components, as can occur through inefficient separation or degradation of the biological particles of interest. Perhaps well-known and exemplary of the shortcomings associated with finding rare and difficult to identify analytes is the identification of circulating tumor cells (CTC), as explained in more detail at https:en.wikipedia.org/wiki/Circulating_tumor_cell on the Wikipedia website.
For example, microfluidic flow through design, as shown in FIG. 1, is well-recognized as being inefficient and slow. As discussed above, the typical microfluidic design involves a layout 10 defining a pathway 12 through which a fluid sample flows, as indicated by the direction A. Multiple analytes 14 present in a fluid sample, as well as target analytes 16 sought to be detected, are caused to flow past barriers, flow-channels, grids, and the like, represented as 18, whereby the physical barriers provided by such structure 18 are operative to selectively control the rate and position by which the analytes flow through the system. The best method takes 15 hours to process 7.5 mL of whole blood and yields only a 40% recovery. These methods are not suitable for commercial scaling applications such as screening patients for cancer cells. See, e.g., Miyamoto, D. T., et. al. Nat. Rev. Clin. Oncol. 11, 401-412 (2014). “Studies have shown that there are several advantages to using the HB-Chip over the CTC chip to capture circulating tumor cells. First, the HB-Chip has the capacity to filter blood at higher flow rates than the CTC chip while still maintaining efficiency. At low flow rates, about 0.12 mL/hr, the cell capture efficiency for the HB-Chip averages 79%, while flat chamber devices, like the CTC chip, average 29%. When flow rates reach up to 0.48 mL/hr, the HB-chip manages a cell capture efficiency of more than 40%, while the average efficiency for a CTC chip at this rate is around 8%.”
With respect to the drawbacks associated with magnetic separation techniques, there is shown in FIGS. 2-5 how such magnetic separation techniques are ineffectual to effectively draw out and isolate the target biological particle/macromolecule of interest. As referenced above, the ability to couple paramagnetic particles to target analytes of interest are well-known in the art, and as shown FIG. 2, there is depicted a container 20 with a bulk liquid specimen containing multiple analytes 22 and target analyte binding paramagnetic particles 24. Based on the ability to attract the paramagnetic particles via the application of a magnetic field thus serves as a basis for separating out such particles along with the bound analytes of interest; however, the prior art application of magnetic fields to such system is sub-optimal.
With respect to FIG. 3, which utilizes a small magnet 30 producing a small static magnetic field 32 about a select area of the sample collection device 20, it is readily recognizable that focusing the target into a small area using a small static magnetic field is ineffective. Due to the exponential loss of magnetic field strength with distance from magnetic source, a small magnetic source, such as 30, cannot project a sufficient magnetic field 32 to penetrate the entire specimen. Such an approach relies on random diffusion of the target into attractive forces of the magnetic field 32. This approach is further disadvantaged by the necessity to transfer the target to a much smaller reaction vessel for subsequent analysis.
Alternatively, as shown in FIG. 4, the use of a larger magnet 34 to produce a correspondingly larger magnetic field 36 that penetrates the entire volume of the specimen inherently draws target to a proportionately large capture area that complicates or thwarts attempts to consolidate rare targets such as 24 into a volume suitable for analysis chambers. In this regard, the magnetic field 36 and subsequent zone of capture has too great of a surface area to effectively isolate and concentrate the sought-after analyte 24.
Referring now to FIG. 5 there is shown a further magnetic separation technique whereby a magnetic source 40 is immersed into a specimen in order to increase the efficiency of the magnetic field 42 to attract the target analyte (i.e., biological macromolecule or cell) 24; however, the challenge of transferring the target 24 from the magnet 40 and resuspending the target analyte in a much smaller volume container can result in loss, damage and/or degradation of the target biological particle. To address this shortcoming, the prior art has relied upon elaborate mechanical sheath-type devices and methods whereby a sheath is placed between the magnetic source and the target such that said target is attached to the surface of the sheath which allows the magnet to be removed. Inevitably, this approach results in the target analyte being spread over a large surface area, which in turn requires removal of target analyte from the sheath using a wash volume, thus again creating a dilute solution of the analyte.
Even after the most effective techniques are used to enrich or maximize the concentration of the population of biological particles of interest in a given sample, the volume of the sample is still oftentimes far too large to allow for accurate and thorough investigation as to whether the particle is present, and much less to what degree. For example, from a 7.5 ml specimen sample, in order to perform PCR (i.e., polymerase chain reaction for analysis of short sequences of DNA or RNA), a 60× to 1500× volume reduction is required insofar as PCR is well-known to have a working volume 0.005 to 0.125 ml, and a maximum volume of 0.20 ml. Similarly, microscope slides typically have a working volume of approximately 0.002 to 0.007 mL and a maximum volume of 0.020 mL, and would require a 1071× to 3750× volume reduction of a 7.5 ml sample in order to reach a manageable volume. Still further, for tests performed in microwells, each microwell typically has a working volume of 0.075 to 0.200 ml, and a maximum volume of 0.36 ml. A 37.5× to 100× volume reduction would thus be required to make the sample suitable.
Accordingly, there is a substantial need in the art for systems and methods that can effectively detect, separate and isolate target analytes of interest, and in particular biological macromolecules and cells of interest that may be present in very low quantities whereby a large, bulk specimen or sample is both reduced in volume to an acceptable working volume and the target analyte of interest being concentrated or enriched therein. There is a further need for such systems and methods that can utilize magnetic and other enrichment methods so as to increase the concentration or presence of a target analyte of interest in a sample that is reduced from a first large or bulk volume to an acceptable working volume. Such improved systems and methods are further preferably of simple design, easy to operate, can produce highly accurate and reproducible results, are relatively inexpensive and time efficient to perform and exceptionally effective in detecting, separating and isolating target analytes of interest in a manner that minimizes sample loss and/or potential contamination or degradation of the target analyte.